HIV-1 gp41 by a common immunological epitope induces increased levels of antibodies against human interferon-beta in HIV-1 positive individuals

Based on our finding that a similar epitope exists between human IFN-beta (aa128-134) and HIV-1 gp41 (aa586-595), we examined 20 sera from healthy and 20 from HIV-1 infected individuals for IFN-beta antibody levels by ELISA. The levels of anti-IFN-beta antibody in sera from HIV-infected individuals were increased by about 160% in comparison with HIV-negative. We affinity-purified anti-gp41 antibodies from sera of HIV-1-infected individuals using rsgp41-sepharose column. One of three antibodies could recognize human IFN-beta in comparison with antibodies from serum of a healthy individual. A mouse antiserum to human IFN-beta recognized rsgp41 (recombinant soluble gp41 Env amino acid 539-684), while the normal mouse serum (pre-immune serum) did not bind to rspg41. These results indicate that a common immunological epitope exists between human IFN-beta and HIV-1 gp41. The sequence-similarity suggests that this common immunological epitope may be located in the region aa128-134 of human IFN-beta and the immunosuppressive domain (aa583-599) of HIV-1 gp41. The increased levels of antibodies against interferon-beta in HIV-1 positive individuals may be explained by a common immunological epitope on human IFN-beta and HIV-1 gp41.     

Modification of delivery system enhances MHC nonrestricted immunogenicity of V3 loop region of HIV-1 gp120

A successful peptide vaccine for AIDS is desired to elicit T-helper and cytotoxic T lymphocyte responses besides neutralizing antibodies. The V3 loop peptide of HIV-1 has been shown to contain the principal neutralizing domain, one of the most immunodominant regions, having both B-cell and T-cell determinants. In this study, the tip of the V3 loop region was mutated from GPGR to GPGQ based on the sequence of Indian isolates (CKRKIHIGPGQAFYT). To further enhance the immunogenicity of this epitope, two delivery systems of immune stimulating complexes (ISCOMs) and liposomes were used to incorporate the peptide. Mice of differing haplotypes, H-2b, H-2d, H-2k and H-2s, showed no MHC restriction when immunized with these formulations. The IgG levels as assessed by ELISA were found to be significantly higher (P < 0.05 to P < 0.001) for even five-fold lower doses of the peptide in ISCOMs and liposomes as compared to the conventional alum-based preparation. The major subtype elicited was IgG2a/IgG2b, suggestive of a Th1-like response for all the formulations. Thus, it would appear that the same peptide incorporated in ISCOMs and liposomes selects a Th1 response and may therefore be important not only for neutralization but also for virus clearance.     

Induction of HIV-1 IIIb neutralizing antibodies in BALB/c mice by a chimaeric peptide consisting of a T-helper cell epitope of Semliki Forest virus and a B-cell epitope of HIV

A colinearly synthesized peptide consisting of a H-2d restricted T-helper cell epitope of Semliki Forest virus (SFV) and triple repeats of sequence GPGRAF, derived from the V3 domain of HIV-1 strains, was used to immunize BALB/c (H-2d) mice. Pepscan analysis of sera from peptide-immunized mice revealed that the chimaeric peptide GREKFTIRPHYGKEIGPGRAFGPGRAFGPGRAF contains three distinct antibody-reactive sequences GREKFTIR, PHYGKEI and GPGRAF. The chimaeric peptide evoked HIV-1 IIIb neutralizing antibodies in serum as measured in vitro by reduction of syncytia formation and reduction of p24 production as well. So, the T-helper cell epitope of SFV provided help to a small linear neutralization epitope of HIV-1 strains. Interestingly, the T-helper cell epitope alone might induce antibodies cross-reactive with HIV-1 IIIb specific peptide GPGRAFVTIGK which shows some homology (residues underlined) with the antibody-reactive sequence GREKTIR of SFV.     

Study of the V3 loop as a target epitope for antibodies involved in the neutralization of primary isolates versus T-cell-line-adapted strains of human immunodeficiency virus type 1

Previous studies characterized the third variable (V3) loop of the envelope gp120 as the principal neutralizing determinant for laboratory T-cell-line-adapted (TCLA) strains of human immunodeficiency virus type 1 (HIV-1). However, primary viruses isolated from infected individuals are more refractory to neutralization than TCLA strains, suggesting that qualitatively different neutralizing antibodies may be involved. In this study, we investigated whether the V3 loop constitutes a linear target epitope for antibodies neutralizing primary isolates. By using peptides representative of the V3 regions of various primary isolates, an early, relatively specific and persistent antibody response was detected in sera from HIV-infected patients. To assess the relationship between these antibodies and neutralization, the same peptides were used in competition and depletion experiments. Addition of homologous V3 peptides led to a competitive inhibition in the neutralization of the TCLA strain HIVMN/MT-4 but had no effect on the neutralization of the autologous primary isolate. Similarly, the removal of antibodies that bind to linear V3 epitopes resulted in a loss of HIVMN/MT-4 neutralization, whereas no decrease in the autologous neutralization was measured. The different roles of V3-specific antibodies according to the virus considered were thereby brought to light. This confirmed the involvement of V3 antibodies in the neutralization of a TCLA strain but emphasized a more pronounced contribution of either conformational epitopes or epitopes outside the V3 loop as targets for antibodies neutralizing primary HIV-1 isolates. This result underlines the need to focus on new vaccinal immunogens with epitopes able to induce broadly reactive and efficient antibodies that neutralize a wide range of primary HIV-1 isolates.     

Identification of the V1 region as a linear neutralizing epitope of the simian immunodeficiency virus SIVmac envelope glycoprotein

The sequence variability of viral structure polypeptides has been associated with immune escape mechanisms. The V1 region of simian immunodeficiency virus (SIV) is a highly variable region of the SIVmac env gene. Here, we describe the V1 region as a linear neutralizing epitope. V1 region-specific neutralizing antibodies (NAb) were first demonstrated in a rabbit infected with a recombinant vaccinia virus carrying the env gene of human immunodeficiency virus type 2 strain ben (HIV-2ben). Since we detected in this animal V1 region-specific NAb that were able to neutralize not only human immunodeficiency virus type 2 but also SIVmac32H, we investigated whether a similar immune response is evoked in macaques (Macaca mulatta) either infected with SIVmac or immunized with the external glycoprotein (gp130) of the same virus. Distinctly lower NAb titers were found in the SIVmac-infected animals than in the gp130-immunized macaques. Since the NAb titers in both groups were high enough for competition experiments, we used five overlapping peptides encompassing the whole V1 region for a detailed identification of the epitope. In each of the 12 macaques investigated, we detected a high level of NAb reacting with at least one peptide located in the central part of the V1 region. The relatively high degree of divergence, especially within the central part of the V1 region, which characterized the evolution of the retroviral sequences from the original inoculum in the infected macaques suggests the development of escape mutants. Furthermore, 3 of 12 animals developed NAb directed against the amino-terminal end of the V1 region epitope. Sequence analysis, however, revealed relatively low levels of genetic drift and genetic variability within this part of the V1 region. The induction of V1 env-specific NAb not only in gp130-immunized macaques but also in SIVmac-infected animals in combination with the increased genetic variability of this region in vivo indicates a marked biological significance of this epitope for the virus.     

Stereochemical analysis of the antigenic tip of the V3 loop peptide of HIV-1 gp120

A novel multiple turn conformation has been observed for a segment GPGRAFY in the crystal structure of a complex of HIV-1 gp120 V3 loop peptide with the Fab fragment of a neutralizing antibody [Ghiara et al. (1994) Science 264, 82-85]. A structural motif has been defined for the peptide segment, employing idealized backbone conformations characterized by ranges of virtual C alpha torsion angles and bond angles. A search of 122 high-resolution protein crystal structures has permitted identification of 24 examples of similar structural motifs. Two major conformational families have been identified, which differ primarily in the conformation at residue 3. The observed conformation at residue 3 in family 1 is left-handed helical (alpha L) and that in family 2 is right-handed helical (alpha R). Of the 10 examples in family 1, 9 examples have Gly residues at position 3. Of the 12 examples in family 2, 7 examples have Asn/Asp at position 3. Computer modeling of the V3 loop tip sequence using the two backbone conformational families as starting points leads to minimum-energy conformations in which antigenically important side-chains occupy similar spatial arrangements. This stereochemical analysis of the V3 loop tip sequence suggests a rational basis for the design of synthetic analog peptides for use as viral antagonists or synthetic antigens.     

Detection of neutralizing antibodies against human T-cell leukemia virus type 1 using a cell-free infection system and polymerase chain reaction

We have developed a cell-free infection system to titrate neutralizing antibodies against human T-cell leukemia virus type 1 (HTLV-1) using the polymerase chain reaction (PCR). S+L-CCC (8C) feline kidney or U-251 MG human glioma cells were infected with a cell-free culture supernatant derived from HTLV-1-infected c77 feline cells. DNA was extracted from 8C or U-251 MG cells after incubation for 24 hr and amplified by PCR. The c77 cell supernatant gave discrete bands, whereas those of HTLV-1-positive T cells did not. When the inocula were treated with HTLV-1 antibody-positive human sera or the monoclonal or polyclonal antibody against the peptide 190-199 of HTLV-1 envelope protein gp46, the subsequent formation of HTLV-1 proviral DNA was inhibited. We determined the titers of neutralizing antibodies by densitometrically scanning the intensity of the PCR bands. These titers correlated well with those determined by the plaque assay using a pseudotype of vesicular stomatitis virus bearing the envelope antigens of HTLV-1. At high serum concentrations, many seronegative samples markedly inhibited the plating of the HTLV-1 pseudotype whereas they barely affected results obtained by PCR. Thus, the c77-PCR system can detect neutralizing antibodies against HTLV-1 even at low titers.     

A neutralizing monoclonal antibody previously mapped exclusively on human immunodeficiency virus type 1 gp41 recognizes an epitope in p17 sharing the core sequence IEEE

We report here that a human immunodeficiency virus type 1 (HIV-1)-specific neutralizing monoclonal antibody (MAb 1575) mapped to the conserved putative intracellular region from amino acid residues 735 to 752 (735-752 region) of gp41 also recognizes a region in an extracellular portion of p17. Both epitopes have a core recognition sequence (IEEE) in a nonhomologous context. The IEEE motif found in HIV-1 p17 is located in a region known as HGP-30 (residues 86 to 115) which has been previously associated with virus neutralization, cytotoxic T lymphocyte activity, and mother-to-child transmission. An analysis of available gp41 and p17 sequences demonstrates that in these regions both IEEE sequences are highly conserved in different HIV-1 clades. The presence of the IEEE epitope in p17 allows us to explain some unexpected neutralizing characteristics of MAb 1575. In addition, the gp41 735-752 region has been previously reported both in intra- and extracellular locations. Our results suggest that the extracellular location was the result of cross-reactivity with p17.     

The ability of HIV type 1 to use CCR-3 as a coreceptor is controlled by envelope V1/V2 sequences acting in conjunction with a CCR-5 tropic V3 loop

Although infection by primary HIV type 1 (HIV-1) isolates normally requires the functional interaction of the viral envelope protein with both CD4 and the CCR-5 coreceptor, a subset of such isolates also are able to use the distinct CCR-3 receptor. By analyzing the ability of a series of wild-type and chimeric HIV-1 envelope proteins to mediate CCR-3-dependent infection, we have determined that CCR-3 tropism maps to the V1 and V2 variable region of envelope. Although substitution of the V1/V2 region of a CCR-3 tropic envelope into the context of a CCR-5 tropic envelope is both necessary and sufficient to confer CCR-3 tropism, this same substitution has no phenotypic effect when inserted into a CXCR-4 tropic HIV-1 envelope context. However, this latter chimera acquires both CCR-3 and CCR-5 tropism when a CCR-5 tropic V3 loop sequence also is introduced. These data demonstrate that the V1/2 region of envelope can, like the V3 loop region, encode a particular coreceptor requirement and suggest that a functional envelope:CCR-3 interaction may depend on the cooperative interaction of CCR-3 with both the V1/V2 and the V3 region of envelope.     

Evaluation of a western blot system (DAVIH-BLOT) for the confirmation of HIV-1 antibodies]

Matrix metalloproteinases (MMPs), a sort of important enzymes involved in extracellular matrix metabolism, play critical roles in the process of tissues remodeling, wound healing and metastasis of tumors. Dot blot and in situ hybridization were used in this study to detect the expression and localization of MMP-9, an important proteolytic enzyme implicated in bone resorption in bone tissues. The results showed that the level of MMP-9 mRNA expression in osteoporotic bone tissues was significantly higher than that in normal control group and the cell types that expressed MMP-9 mRNA included mono- and multi-nuclear osteoclasts and some lining cells on the surface of bone matrix. It was suggested that MMP-9 play a key role in the development of bone loss in osteoporosis.     

A peptide mimic of a protective epitope of respiratory syncytial virus selected from a combinatorial library induces virus-neutralizing antibodies and reduces viral load in vivo

Respiratory syncytial virus (RSV) is the most important cause of bronchiolitis and pneumonia in infants and young children worldwide. As yet, there is no effective vaccine against RSV infection, and previous attempts to develop a formalin-inactivated vaccine resulted in exacerbated disease in recipients subsequently exposed to the virus. In the work described here, a combinatorial solid-phase peptide library was screened with a protective monoclonal antibody (MAb 19) to identify peptide mimics (mimotopes) of a conserved and conformationally-determined epitope of RSV fusion (F) protein. Two sequences identified (S1 [HWYISKPQ] and S2 [HWYDAEVL]) reacted specifically with MAb 19 when they were presented as solid-phase peptides. Furthermore, after amino acid substitution analyses, three sequences derived from S1 (S1S [HWSISKPQ], S1K [KWYISKPQ], and S1P [HPYISKPQ]), presented as multiple antigen peptides (MAPs), also showed strong reactivity with MAb 19. The affinity constants of the binding of MAb 19, determined by surface plasmon resonance analyses, were 1.19 x 10(9) and 4.93 x 10(9) M(-1) for S1 and S1S, respectively. Immunization of BALB/c mice with these mimotopes, presented as MAPs, resulted in the induction of anti-peptide antibodies that inhibited the binding of MAb 19 to RSV and neutralized viral infection in vitro, with titers equivalent to those in sera from RSV-infected animals. Following RSV challenge of S1S mimotope-immunized mice, a 98.7% reduction in the titer of virus in the lungs was observed. Furthermore, there was a greatly reduced cell infiltration in the lungs of immunized mice compared to that in controls. These results indicate the potential of peptide mimotopes to protect against RSV infection without exacerbating pulmonary pathology.     

Sera from HIV-1 infected individuals in all stages of disease preferentially recognize the V3 loop of the prototypic macrophage-tropic glycoprotein gp120 ADA

The outer membrane glycoprotein gp120 and the transmembrane glycoprotein gp41 are predominant targets of the humoral immune response to infection by human immunodeficiency virus type 1. The third hypervariable region (V3 loop) is the principal neutralizing domain and is the primary target of neutralizing antibodies directed against the envelope proteins of HIV-1. The V3 loop is also the major determinant for HIV-1 cell-specific tropism. To further characterize the humoral immune response directed against the gp120 envelope proteins, we expressed two prototypic gp120 envelope proteins (LAI/HXB2 and ADA) and chimeric gp120 envelope proteins in stable transfected Drosophila melanogaster Schneider 2 cells. Sera from four infected adults over the course of infection [McNearney et al. (1992) Proc. natn. Acad. Sci. U.S.A. 89, p. 10,242] were assayed for reactivity with the respective envelope proteins. Sera obtained at early stages preferentially recognized the gp120 envelope protein ADA, whereas in later stages of infection the sera showed diminished reactivity with both gp120 LAI/HXB2 and gp120 ADA. Chimeric envelope proteins revealed that the humoral response was directed primarily against the V3 loop of gp120 ADA. Furthermore, 22 sera from HIV-1 infected individuals in different stages of the disease were tested. Reactivity of sera with the gp120 envelope protein ADA was seven-fold higher than with the gp120 envelope protein LAI/HXB2. Our results suggest that the humoral immune response is preferentially elicited against the V3 loop of the prototypic macrophage-tropic gp120 envelope protein ADA.     

Stepwise transformation of a cholera toxin and a p24 (HIV-1) epitope into D-peptide analogs

BACKGROUND: The evidence suggests that personality traits are hierarchically organized with more specific or lower-order traits combining to form more generalized higher-order traits. Agreement exists across studies regarding the lower-order traits that delineate personality disorder but not the higher-order traits. This study seeks to identify the higher-order structure of personality disorder by examining the phenotypic and genetic structures underlying lower-order traits. METHODS: Eighteen lower-order traits were assessed using the Dimensional Assessment of Personality Disorder-Basic Questionnaire in samples of 656 personality disordered patients, 939 general population subjects, and a volunteer sample of 686 twin pairs. RESULTS: Principal components analysis yielded 4 components, labeled Emotional Dysregulation, Dissocial Behavior, Inhibitedness, and Compulsivity, that were similar across the 3 samples. Multivariate genetic analyses also yielded 4 genetic and environmental factors that were remarkably similar to the phenotypic factors. Analysis of the residual heritability of the lower-order traits when the effects of the higher-order factors were removed revealed a substantial residual heritable component for 12 of the 18 traits. CONCLUSIONS: The results support the following conclusions. First, the stable structure of traits across clinical and nonclinical samples is consistent with dimensional representations of personality disorders. Second, the higher-order traits of personality disorder strongly resemble dimensions of normal personality. This implies that a dimensional classification should be compatible with normative personality. Third, the residual heritability of the lower-order traits suggests that the personality phenotypes are based on a large number of specific genetic components.     

SPC3, a V3 loop-derived synthetic peptide inhibitor of HIV-1 infection, binds to cell surface glycosphingolipids

Synthetic multibranched peptides derived from the V3 domain of human immunodeficiency virus type 1 (HIV-1) gp120 inhibit HIV-1 entry into CD4+ and CD4- cells by two distinct mechanisms: competitive inhibition of HIV-1 binding to CD4-/GalCer+ colon cells and postbinding inhibition of HIV-1 fusion with CD4+ lymphocytes. In the present study, we have characterized the cellular binding sites for the V3 peptide SPC3, which possesses eight V3 consensus motifs GPGRAF radially branched on a neutral polyLys core matrix. These binding sites are glycosphingolipids that share a common structural determinant, i.e., a terminal galactose residue with a free hydroxyl group in position 4: GalCer/sulfatide on CD4-/GalCer+ colon cells; LacCer and its sialosyl derivatives GM3 and GD3 on CD4+ human lymphocytes. These data suggest that the V3 peptide binds to the GalCer/sulfatide receptor for HIV-1 gp120 on HT-29 cells and thus acts as a competitive inhibitor of virus binding to these CD4- cells, in full agreement with previously published virological data. In contrast, SPC3 does not bind to the CD4 receptor, in agreement with the data showing that the peptide inhibits HIV-1 infection of CD4+ cells by acting at a postattachment step. The binding of SPC3 to LacCer, GM3, and GD3, expressed by CD4+ lymphocytes, suggests a role for these glycosphingolipids in the fusion process between the viral envelope and the plasma membrane of CD4+ cells. Since the multivalent peptide can theoretically bind to several of these glycosphingolipids, we hypothesize that the resulting cross-linking of membrane components may affect the fluidity of the plasma membrane and/or membrane curvature, altering the virus-cell fusion mechanism.